We have developed rVSVs expressing the glycoproteins (GP) of representative isolates of all species of Ebola virus: Sudan ebolavirus (SEBOV), Zaire ebolavirus (ZEBOV), Cote d'Ivoire ebolavirus (CIEBOV), Bundibugyo ebolavirus (BEBOV) and Reston ebolavirus (REBOV). Additionally, we generated rVSVs expressing the GPs of two isolates of Marburg virus: Lake Victoria marburgvirus isolate Musoke (MARVmus) and Angola (MARVang). Mainly the rVSV/ZEBOVgp, rVSV/SEBOVgp and rVSV/MARVmusGP vaccine vectors have been extensively characterized in cell culture and their protective efficacy has been evaluated in animal models against homologous challenges (Hoenen et al. 2012; Falzarano & Feldmann, 2013). Recently, we were able to demonstrate the protective efficacy of rVSV/BEBOVgp and rVSV/MARVangGP against homologous challenge in nonhuman primates (NHPs) (PLoS Negl Trop Dis, in revision). In an effort to decipher the mechanism of protection of the rVSV vaccine vector against ZEBOV, we were able to demonstrate in NHPs that antibodies specific to the viral antigen play a critical role (Marzi et al., 2013). Ongoing work is investigating the mechanism of protection of the rVSV vaccine vector against MARV. Defining the mechanism(s) and correlate(s) of protection will be milestones for moving the platform into clinical trials. Cross-protection among the different Ebola virus species and even Marburg virus is an important consideration and has been difficult to achieve due to relatively high genetic and antigenic variability among genera in particular, but also among species within a single genus, and the general lack of cross-protective antibodies even among species. In this regard we have previously performed a proof-of-concept study using a single-injection protocol with a blended vaccine including rVSV/SEBOVgp, rVSV/ZEBOVgp and rVSV/MARVmusGP to see if a cross-protective vaccine could be developed against four human pathogenic filoviruses endemic in Central Africa. Challenge was performed four weeks after immunization with MARVmus, SEBOV, ZEBOV and CIEBOV resulting in protection against homologous challenges as well as a heterologous challenge (CIEBOV) indicating that cross-protective vaccines are feasible (Hoenen et al. 2012; Falzarano & Feldmann, 2013). More recently, we have performed another proof-of-concept study in which we evaluated cross-protection following immunization with a single vaccine vector (rVSV/ZEBOVgp or rVSV/CIEBOVgp). A single vaccination with the rVSV/ZEBOVgp provided cross-protection (75% survival) against a subsequent heterologous BEBOV challenge, whereas vaccination with the rVSV/CIEBOVgp resulted in no protection. This demonstrates that monovalent rVSV-based vaccines may be useful against a newly emerging species; however, heterologous protection across species remains challenging and may depend on enhancing the immune responses either through booster immunizations or through the inclusion of multiple immunogens (Hoenen et al. 2012; Falzarano & Feldmann, 2013). Overall, we can conclude that single monovalent rVSV vaccine vectors can provide partial cross-protection in cases of challenge viruses that are genetically more closely related. One approach to overcome this limitation is the use of blended monovalent rVSV vaccine vectors, which provide broader cross-protection against homologous and partial protection against certain heterologous challenges. Another approach to overcome the limitations in cross-protection is the use of multivalent rVSV vaccine vectors. In a proof-of-concept study protection against ZEBOV and Andes virus (ANDV) was demonstrated using a single rVSV vector expressing both the ZEBOVgp and ANDV glycoprotein in the Syrian hamster model. This data showed that bivalent rVSV vectors are a feasible approach to vaccination against multiple pathogens. Further, this study demonstrated that the Syrian hamster is an adequate model to study rVSV-mediated protection (Hoenen et al. 2012; Falzarano & Feldmann, 2013). Based on the results described above, we have in the past fiscal year generated bivalent and trivalent rVSV vectors expressing two or three different filovirus GPs, one as a transmembrane protein (replacing the VSV glycoprotein) and one or two as soluble proteins that will be secreted during vector replication. Recovery of these recombinant vaccine viruses and in vitro characterization are ongoing. Efficacy testing of these vectors will be performed initially using rodent models, mainly the Syrian hamster. The most promising vaccine vectors will be moved into efficacy testing in the nonhuman primate model for filovirus infections.